Enterococcus faecalis is a major cause of hospital-acquired infections and also plays a key role in the dissemination of antibiotic resistance and virulence genes to other bacterial pathogens. This application seeks continued support for basic studies of a unique peptide-based signaling system that allows cells carrying an antibiotic resistance/virulence plasmid called pCF10 to regulate expression of genes involved in resistance transfer and virulence. The key players in the signaling system include the cCF10 peptide pheromone (signal molecule) and the iCF10 peptide pheromone inhibitor which antagonizes cCF10. The receptor for these peptides is a transcription factor called PrgX, which regulates expression of the pCF10 prgQ operon encoding the virulence and plasmid transfer genes. The pheromone response machinery comprises a sensitive and robust biological switch, where multiple regulatory circuits act in concert to amplify the direct response to pheromone, and to enable the host cell to sense different kinds of environmental cues. Post-transcriptional regulatory mechanisms involving novel small RNAs also play a major role in the pheromone response. The proposed studies are focused on elucidating the quantitative effects mechanistic aspects of various regulatory circuits, and on determining the mechanisms by which the pheromone-induced state can be returned to basal levels of expression following transfer of the plasmid to a new host. This work will reveal insights into regulation of microbial virulence by cell-cell signaling, applicable to many organisms, and may also facilitate new strategies for development of antimicrobial agents.